Plasmid-encoded tetracycline resistance is responsible for most known resistance to this antibiotic. In many organisms, resistance is regulated: tetracycline induced increased resistance to itself accompanied by decreased uptake of the drug. We have demonstrated induced synthesis of an inner membrane protein, TET protein and, presumably, expression of much of the tetracycline resistance determinant. Our studies link resistance both to altered transport of the drug and to an intracellular "factor" which counteracts tetracycline activity. We shall focus our studies in four areas: 1) mechanism of altered transport and the cellular location of transport systems; 2) biochemical and functional analysis of the membrane TET protein with emphasis on its role in tetracycline resistance; 3) characterization of the intracellular inhibition of tetracycline activity; and 4) role of chromosomal gene products in expression of plasmid-mediated resistance. In studying the transport systems, we shall use whole cells, spheroplasts, membrane vesicles and toluene-treated cells. We shall also assess equilibrium binding of tetracycline to different cellular components. In analyzing TET protein we shall use purified TET protein to convert sensitive vesicles into resistant vesicles and we shall identify any changes in TET protein encoded by mutated resistances by use of R plasmid-in-minicell system and lambda bearing Tn10 mutations. In characterizing intracellular inhibition, we shall examine tetracycline sensitivity of total protein synthesis and of the specific tetracycline-sensitive steps using different in vitro systems derived from sensitive and resistant cell extracts. In the above studies and in those examining cellular influence of plasmid-mediated resistance, both plasmid and chromosomal mutants affecting tetracycline resistance will be used.